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Binding of -synuclein oligomers to Cx32 facilitates protein uptake and transfer in neurons and oligodendrocytes
Linköping University, Department of Clinical and Experimental Medicine, Divison of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Divison of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0002-1273-6731
Univ Hosp Erlangen, Germany.
Karolinska Inst, Sweden.
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2019 (English)In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 138, no 1, p. 23-47Article in journal (Refereed) Published
Abstract [en]

The intercellular transfer of alpha-synuclein (-syn) has been implicated in the progression of Parkinsons disease (PD) and multiple system atrophy (MSA). The cellular mechanisms underlying this process are now beginning to be elucidated. In this study, we demonstrate that the gap junction protein connexin-32 (Cx32) is centrally involved in the preferential uptake of -syn oligomeric assemblies (o-syn) in neurons and oligodendrocytes. In vitro, we demonstrate a clear correlation between Cx32 expression and o-syn uptake. Pharmacological and genetic strategies targeting Cx32 successfully blocked o-syn uptake. In cellular and transgenic mice modeling PD and MSA, we observed significant upregulation of Cx32 which correlates with -syn accumulation. Notably, we could alsodemonstrate a direct interaction between -syn and Cx32 in two out of four human PD cases that was absent in all four age-matched controls. These data are suggestive of a link between Cx32 and PD pathophysiology. Collectively, our results provide compelling evidence for Cx32 as a novel target for therapeutic intervention in PD and related -synucleinopathies.

Place, publisher, year, edition, pages
SPRINGER , 2019. Vol. 138, no 1, p. 23-47
Keywords [en]
Parkinsons disease (PD); Multiple system atrophy (MSA); Alzheimers disease (AD); Cell-to-cell transfer; Prion-like transfer; Gap junction proteins; Cx32; GJB1; alpha-Synuclein (-syn)
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:liu:diva-158850DOI: 10.1007/s00401-019-02007-xISI: 000471708700002PubMedID: 30976973OAI: oai:DiVA.org:liu-158850DiVA, id: diva2:1337646
Note

Funding Agencies|Swedish Research Council [523-2013-2735]; Deutsche Forschungsgemeinschaft (DFG) [GRK2162]

Available from: 2019-07-16 Created: 2019-07-16 Last updated: 2019-10-14
In thesis
1. Investigation of the intercellular transmission of α-synuclein, amyloid-β and TDP-43
Open this publication in new window or tab >>Investigation of the intercellular transmission of α-synuclein, amyloid-β and TDP-43
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS) are disorders characterized by the progressive deposition of proteinaceous inclusions throughout the brain in a predictable manner. Each disease is described by the involvement of different misfolded and aggregated proteins (AD, amyloid-β and tau; PD, α-synuclein; ALS and FTLD, TDP-43) that spread between anatomically connected brain regions, causing cell death in previously healthy regions. Disease progresses as these aggregated proteins spread throughout the brain in a prion-like fashion. Oligomeric forms of these proteins (aggregates comprising of ≈3-30 individual proteins) are thought to be the most relevant to disease, as they are capable of prion-like propagation and can cause cellular toxicity. The work in this thesis aims to elucidate the mechanisms by which different neurodegenerative disease related proteins (amyloid-β, α-synuclein and TDP-43) are taken up and transferred between cells, and the effects exerted by these proteins on downstream cells.

Paper I examined the uptake and cell to cell transmission of oligomeric α-synuclein (α-syn). Using a 3D co-culture model, we determined that α-syn (monomeric, oligomeric and fibrillar assemblies) were readily taken up and transferred between neuron-like cells, and that this transfer was mediated by an endosomal/lysosomal mechanism. It was also determined that larger α-syn assemblies (oligomers and fibrils) were found in donor and acceptor cells more frequently than monomeric α-syn, which we speculate is a due to the larger aggregates’ resistance to cellular proteases.

In Paper II, we identified a novel mechanism for the uptake of oligomeric proteins, in the discovery that the gap junction channel protein connexin 32 mediates the uptake of α-syn oligomers in a preferential manner. Gap junction proteins act as a means of communication between adjacent cells, forming a transmembrane pore to facilitate the passage of small molecules. Here, we determined that connexin 32 drives the preferential uptake of oligomeric α-syn relative to monomeric and fibrillar α-syn. This system was not exclusive to α-syn however, as the preferential uptake of oligomeric amyloid-β (Aβ) was also observed. In addition to the uptake of oligomers, we observed that increased α-syn expression elicited the increased expression of connexin 32, in a positive feedback mechanism. When connexin 32 was inhibited pharmacologically or knocked out using CRISPR/Cas9, the preferential uptake of oligomers was abolished. These phenomena were also observed in oligodendrocytes (the accumulation of oligomeric α-syn in oligodendrocytes is a hallmark of Multiple Systems Atrophy), three different mouse models of α-syn overexpression, as well as in post-mortem human tissues.

Paper III undertook the investigation of cell to cell transfer of TDP-43. Although it was recently confirmed that TDP-43 propagates throughout the brain in a prion-like fashion, it remains unclear how post-translational modifications of TDP-43 affect its propensity to be transferred between cells. This leaves a gap in the understanding of how TDP-43 proteinopathies progress, as post-translationally modified TDP-43 is understood to be critical to pathogenesis. To study this, we generated several TDP-43 cell lines, expressing full-length TDP-43 or C-/N-terminally truncated fragments, known contributors to TDP-43 proteinopathies. Using the 3D co-culture model, we determined that preservation of the N-terminus of TDP-43 enhanced its ability to transmit between cells, whereas an intact the C-terminus reduced transfer. Additionally, since we have previously shown that both oligomeric Aβ and α-syn are incorporated into extracellular vesicles (EVs) such as exosomes, and that these EVs can sufficiently mediate the transfer of protein oligomers to downstream cells, we investigated whether this was also true for TDP-43. We demonstrated that full-length TDP-43 and TDP-43 fragments could be found within EVs generated by these cells, but that these EVs were unable to propagate the protein to downstream cells. Instead, the transmission of TDP-43 occurs in a manner dependent upon physical proximity between cells, possibly across the synaptic cleft itself.

Next, we studied the acute effects exerted by oligomeric Aβ upon healthy neurons in order to understand the earliest effects of oligomeric Aβ challenge. In Paper IV, we used iPSC-derived neurons generated from human donors expressing different amyloid-β precursor protein (APP) genes, one harbouring the familial AD-causing V717I London mutation, the other expressing WT APP. After differentiating these cells into functional neurons in vitro, the neurons were challenged with acute exposure to exogenous oligomeric Aβ and analyzed by LC-MS/MS to observe the early effects. By analyzing the proteome and phosphoproteome of these cells, we identified many proteins and phosphoproteins that were up- or down-regulated in response to oligomeric Aβ at this early timepoint. Among these changes, oligomeric Aβ caused the downregulation of TDP-43, heterogeneous nuclear ribonucleoproteins, and coatomer complex I proteins. Conversely, increases were observed in 20S proteasome subunits and vesicle associated proteins VAMP1/2. We also observed the differential phosphorylation of tau at serine 208, indicating that phosphorylation at this residue might be an important early event in tauopathy.

Altogether, the work described in this thesis has provided new understanding as to how different neurodegenerative disease related proteins are taken up and transferred between cells. In doing so, we have identified some of the mechanisms by which this spreading occurs, and that the changes elicited by these toxic oligomeric proteins are rapid and widespread. By learning about these processes, we have identified novel targets that could be used in the development of disease modifying therapeutics.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2019
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1702
Keywords
α-synuclein, amyloid-β, TDP-43, cell to cell transmission
National Category
Neurosciences
Identifiers
urn:nbn:se:liu:diva-160318 (URN)978-91-7519-015-0 (ISBN)
Public defence
2019-11-07, Berzelius, Campus US, Linköping, 09:00 (English)
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Supervisors
Available from: 2019-10-14 Created: 2019-09-26 Last updated: 2019-10-14Bibliographically approved

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